| Cellulose is rich in nature as the content of renewable resources and cheap. According to estimates, The cellulose material in synthetic substances of photosynthesis accounted for about 6%,that can reach 200 million tons on the earth's surface every year.At the moment,the create capacity of dry matter is reach as high as 15 million tons every year in the world. The amount of cellulose and hemicellulose about 850 million tons.So The cellulose is inexhaustible in nature. In the present for the transformation of the decomposition of cellulose research, mainly involves the development of cellulose microorganism resources, cellulose enzyme activity enhancement and optimization of reaction conditions. Cellulase is a kind of very highly active biological catalyst, it is the hydrolysis of cellulolytic enzymes generated a group of glucose non-functional. It consists of three components enzyme synergy:(])endo-1,4-β-D-glucanases (EQC1orCMC), (2)exo-1,4-β-D-glucanases(CBH or Cx),(3) 1,4-β-D-glucosidases (BG).1. This study is using a hot issue in allusion to cellulose. To Trichoderma viride as the research object, Using physical and chemical respectively method for high-yielding cellulose enzyme activity mutagenesis breeding the degradation bacterium. Optimize enzyme production conditions, and study for enzymology character. Use five different mutation methods ,which is including fast neutron, ultraviolet rays, y-rays, diethyl sulfate, sodium nitrite, to Trichoderma viride AS3.3711 for mutagenesis breeding. Usually, after the mutation processing, the lethality and the radiation dosage has a linear positive correlation within the scope of certain. Through the test results:(1) Sodium nitrite mutagenesis:During the fungus levitation liquid join acetic acid buffer fluid,and different concentrations of sodium nitrite solution with 0.1mol/L,0.05 mol/L,0.03 mol/L,0.02 mol/L. Respectively at room temperature heat preservation for 5,10,15,20min. The optimal concentration was 0.1mol/L and the optimal time is 20 min. The enzyme activity of CMC of mutations increased to 1.74 times.(2) Diethyl sulfate mutagensis:The volume ratio of diethyl sulfate and mycete was 1%,0.5%,0.3%,0.2%. In 30℃conditions oscillation processing for 20,30,40,60min. The optimal volume ratio was 1% and the optimal time is 40 min. The enzyme activity of CMC of mutations increased to 2.8 times.(3) Ultraviolet rays mutagensis:In 256nm and 354nm place respectively, the vertical distance to 20,25,30 cm, with irradiation treatment 60,120,180,240,300,360,600 s, The optimal wavelength was 354 nm, the optimal vertical distance was 30 cm and the optimal time was 240s. The enzyme activity of CMC of mutations increased to 2.64 times.(4)γ-rays mutagensis:Use the Radiation dose rate 0.4 Gy/min and the irradiation time with 2.5,3.75,5,6.25, 10min, The enzyme activity of CMC of mutations only increased to 1.29 times.(5) Fast neutron mutagensis:Use the Radiation dose rate 0.002 Gy/s and the irradiation time with 5,10,20, 30,40min, it meant 0.6,1.2,2.4,3.6,4.8Gy, The enzyme activity of carboxymethyl cellulose of Fn10-1 can reach 914.80 U/mL,that increased to 30 times than the primitive strain.Use of Trichoderma viride AS3.3711 for fast neutron irradiation processing, to the hydrolysis circle of mutant strain on the congo red medium, whose diameter of the ratio of the colony, and cellulose enzyme as the basis, to obtain high mutation strain. Through the experiments found that low doses of fast neutron irradiation can greatly improve cellulose enzyme activity. The mutation rate is 14.3% when the irradiation dose for 1.2 Gy. We got the stability good of mutation Fn10-1 by means of screening and genetic stability experiment. The enzyme activity of carboxymethyl cellulose (enzyme activity of CMC) of Fn 10-1 can reach 914.80 U/mL,that increased to 30 times than the primitive strain. The enzyme activity of filter paper (enzyme activity of FPA) of Fn10-1 can reach 633.63 U/mL, that increased to 4 times than the primitive strain.2. Through the ammonium sulfate grading precipitation method, the paper obtained the cellulose enzyme of Fn10-1 and measured this cellulose enzyme characteriztion. The enzymology character experiment including optimal pH value, stability, optimal temperature, thermal stability, activator and inhibitor. The results showed that determined 30% and 80% two saturation for grading precipitation gradient. First, joined solid ammonium sulphate to 30%, static overnighted, got the supernate after the centrifugation. And then joined solid ammonium sulphate to 80% again, static overnighted, got the precipitate after the centrifugation.3.The precipitate was dissolved by phosphate buffer, namely for coarse enzyme liquid, to the enzymology character experiment subsequent. After the enzymology character experiment we got the results showed that its optimal pH value was 7.6, between 5.6 to 7.6, it had higer stability. Its optimal temperature was 70℃, And it had high stability when the temperrature was under 50℃. For this enzyme, Mg2+,Co2+,Na+,Mn2+,Fe2+ and Al3+ had obvious activation function, well, Ba2+,Ag2+,Cu2+ and K+ had inhibitory action on it. |
